How to Send Pathological Specimens?

Dr. Md. Sadequel Islam Talukder
MBBS; M. Phil (Pathology)
Assistant Professor of Pathology
Mymensingh Medical College       
 

• Sending of specimen is the most important step in the pathological investigation.

•  Any mistake at this step renders all the labour fruitless and the test report becomes doubtful or insignificant. 

• Therefore, all necessary precautions must be taken at this step.       

• A properly filled request form should be accompanied by the specimen.      

• The request form should include patients’ identification, brief clinical history and provisional diagnosis.       

• Name of investigation should be written clearly in usual terms, so that, every laboratory receptionist, technologist and pathologist can easily understand.      

• Request form should also include name of specimen, method of collection (if collected by referring doctor) and preservative and anticoagulant (if used).

• Request form should be signed by referring doctor with name and designation.

• It is better to send the patient to the laboratory. If not possible, the referring doctor or his/her assistant or nurse can collect the specimen.   

• Before collecting specimen, all containers must be labelled properly giving full details of patient’s identification.     

• The common laboratory specimens are blood, urine and stool. Special specimens are swab, smear, aspirates, surgical specimen (biopsy) etc.     

• The procedures of collection and sending specimens for pathological investigations are presented here:  

Blood:

Blood sample may be three types:

1. Venous Blood
2.  Arterial Blood
3.  Capillary Blood  

Venous Blood:     

• It is most commonly required blood sample ·       
• Majority of routine tests are performed out.    
• Obtained directly from vein.    
• May be used in three forms: 1. Whole blood 2.    Plasma 3.    Serum    Contents

Whole Blood: Whole blood is collected in different ways for the purpose of different investigations. These are:   a)    Whole blood anticoagulated with dry anticoagulant – used for cell count, film examination, urea, glucose, ammonia, pH etc. b)    Whole blood anticoagulated with liquid anticoagulant – ESR. c)     Whole blood anticoagulated with heparin - used in determination of osmotic fragility and HLA typing. d)    Whole blood anticoagulated by dilution – used for blood culture.       

Plasma:     

• It is obtained by removing all formed elements from anticoagulated blood by centrifugation at 500 rpm for 3-5 minutes.
• Anticoagulant vary depending upon the test required such as coagulation tests, fibrinogen assay etc.  

Serum:  

• It is the fluid left behind after blood has clotted.       
• This is the most common specimen required for chemical and serological tests.    
• A clean plain test tube or bottle is used for blood collection and blood is allowed to clot. The tube is the gently centrifuged to obtain clear serum. Arterial Blood:      
• This sample is occasionally required, particularly for estimation of blood gases.        
• This is obtained directly from a superficial artery like radial, brachial, temporal or femoral arteries.  Contents  

Capillary Blood:    

• This is occasionally required. ·       
• In case of infants and young children, to perform one or two test, blood is obtained from heel pulp, finger pulp or ear lobule. ·     
• Platelet count should never be performed on this sample.  

Urine:

• Urine specimen may be: A.    Random or spot collection B.    Timed (such as 24 hours) collection. Spot urine Collection:      
• For majority of tests a single urine specimen is required.       
• For some tests such as sugar, pregnancy test and for bacteriological tests an early morning first specimen is required, as it is more concentrated.     
• About 100-ml urine is sufficient for routine examination.        
• For bacteriological examination a mid stream specimen is preferred particularly in female. Patient is asked to wash external genitalia with soap and water before voiding urine.  

Timed Urine Collection:    

• For some biochemical analyses, such as 24 hours total protein, 24 hours collection of urine is required. ·       
• Patient should empty his/her bladder in a given hour (say 7 a.m.) and the sample is discarded. All urine voided in next 24 hours should be collected in a container. Next day at 7 a.m. patient must pass urine, which should also be collected. A preservative (such as toluene 10-15 ml) is added to the container before hand.  

Stool:

• Faecal specimens are required for three types of tests: 1.    Parasitological test 2.    Bacteriological test 3.    Chemical test     ·   
• For parasitological test it is best that the patient passes faeces in bedpan so that abnormal looking portion is selected, e.g. portion containing blood and/or mucous, visible worms or segments.        
• If amoebic dysentery is suspected, patient should pass stool in the laboratory to prepare slide while stool is warm and to examine immediately under microscope for vegetative form of amoebae. Immobile amoeba is difficult to identify.        
• For bacteriological examination the stools are collected in transport medium or directly into culture medium. ·       
• For chemical examination container must be clean. Spot specimens are good enough for occult blood. Timed specimens are required for faecal fat estimation.    

Sputum:       

• Sputum is collected in a clean, dry, wide mouthed, leak proof container.        
• Patient is required to cough deeply to produce a sputum specimen. ·       
• Sputum is best collected in the morning when patient wakes up.                                      Contents  

Throat swab:       

• Throat swab is collected by doctor or an experienced person.        
• The affected area is visualised by using tongue depressor and torch. ·       
• The area is rubbed by the swab ·       
• The swab is put back in the container and delivered to the laboratory within 2 hours. ·       
• Patient must not take antibiotic or any antiseptic mouthwash at least 8 hours before taking swab.

Cerebrospinal Fluid (CSF):     

• CSF is collected by physician in hospital or clinic in strict aseptic condition.        
• Specimen is collected in 2-4 ml quantities in 3-4 sterile screw capped bottle, which are serially numbered and must be sent to the laboratory immediately.        
• First bottle is usually contaminated with blood and is not suitable for examination. ·       
• Second bottle is used for routine examination.        
• Third bottle is used for bacterial culture.        
• Fourth bottle is kept in refrigerator undisturbed to see whether pellicle or coagulum forms, if tubercular meningitis is suspected. Otherwise, CSF must never be refrigerated and shall be kept in 37^oC.

Effusion Fluid:                                                                                                                                                                                                 Contents

Effusion fluids are:      1.    Ascitic fluid 2.    Pleural fluid 3.    Pericardial fluid 4.    Synovial fluid 5.    Hydrocele fluid 6.    Aspirates from cyst etc. ·       

• Main objective of fluid examination is to ascertain their transudative or exudative nature, find out causative organism and malignant cells.        
• It is collected by clinician under aseptic condition.        
• Fluid is collected in  3-4 sterile containers. It is better to take a separate specimen in EDTA for cell count.

Bone marrow:        

• Bone marrow aspiration is done by a doctor under aseptic condition.        
• Bone marrow smear can be made directly or after concentration. ·       
• For concentration marrow is collected in EDTA. ·       
• For direct preparation 2-3 slides are placed in a slanting position over a filter paper and marrow is poured at upper end of the slides. Marrow fragments are picked up with edge of a spreader and push –smear is prepared. ·       
• If marrow is collected in EDTA, it is filled in Wintrobe’s tube and centrifuged. Buffy coat along with small amount of plasma is collected by pasture pipette and smear is prepared.    Contents

Skin Scrapping: ·       

• The affected area is cleaned with 70% ethanol. ·       
• Surface or margin of the lesion is scrapped with a sterile scalpel. ·       
• Scrapping material is wrapped in paper and sent to laboratory.  

Pap’s smear: ·       

• Cervical smear or Pap’s smear is collected by gynaecologists or nurses specially trained in it. ·       
• Material is collected with wooden Ayres’s spatula. ·       
• Smear is prepared on one end of a glass slide and fixed in 95% alcohol in koplin jar for 20-30 minutes. ·       
• The slide is wrapped with tissue paper and sent to laboratory.   Biopsy or Surgical Specimen: ·       
• A representative piece of tissue is removed from body to diagnose a disease. It is called biopsy. ·       
• These specimens must retain its gross and microscopic anatomy. To achieve this objective, these specimens are treated with chemicals called fixative and this process is called fixation. ·       
• The following points to be remember during sending a surgical specimen: a)  Container: -        Must be proper size with respect to the specimen -        Should have wide mouth. -        Should have proper lid -        Plastic container is preferred over tin jar. -        Should have smooth and perpendicular wall.                                                   Contents        Specimen is so placed in container that it keeps it shape as it is. b)  Fixative: -        In routine practice 10% formalin is used as fixative. -        It is prepared by dissolving one part of formalin (commercially available 40% formaldehyde) in nine part of water.     -         Specimen for frozen section is sent in normal saline.     c)   Labelling: -        The container must always be labelled in clear hand writing with the help of black marker. -        A request form containing maximum information about the patient must accompany the specimen.          

Cytological Specimen: -       

These specimens are generally sent for malignant cells. -       

After removal from the body the cells will degenerate in 6-8 hours. Degeneration must be prevented by following steps:

•  Specimen should reach the laboratory without delay.
• If there is some delay, equal volume of 50% alcohol should be added as fixative. ·       
• The quantity of fluid should be 20 cc or more. ·       
• Anticoagulant is added if protein is expected high.      

A Model Request forms:

Department of Pathology Histopathology Section Mymensingh Medical College, Mymensingh

Requisition Form

1. Patient’s Name: Mr. Kala Chand
2. Age: 40 yrs    
3. Sex: Male                                                      
4. Reg. No: 12345/03    
5. Ward: 36                                                       Bed: 40     Cabin:                                                           Unit: SU-VI    
6. Date of Admission: 12/3/02                              Date of Operation: 14/3/02 2.
7. Clinical History:

a)    Epigastric pain and vomiting - 4 months
b)    Swelling of left supraclavicular region - 15 days 3.
 

8. Report of relevant investigations:

      a)    Filling defect in stomach in Ba-meal X-ray
    b)    High ESR 4. LPM (if female):

9. Provisional clinical diagnosis: carcinoma of stomach with metastasis to left supraclavicular lymph node

10. Operation note: There was adhesion of lymph node to perinodal tissue

11. Name of specimen: Left supraclavicular lymph node (Incision biopsy)

12. Fixative: 10% Formalin

13. Date: 14/3/02

14. Requested by Dr. Surza Kanta MBBS Assistant Registrar SU-VI